Preparations of scarlet fever toxin for administration by mouth



Patented Feb. 13, 1945 PREPARATIONS OF SCARLET FEVER TOXIN FOR ADLHNISTRATION BY MOUTH George F. Dick and Gladys H. Dick, Evanston, Ill., and Jonathan Wood, Prospect Park, Pa.; said Jonathan E. .Wood assignor to said George F. Dick and said Gladys H. Dick No Drawing.

. Claims.

Application January 2, 1941, sol-m1 No. 372,888

This invention relates to the production and use of a toxin specific to scarlet fever for oral administration. v

The toxin specific to scarlet fever disclosed in the patent to Doctors George F. Dick and Gladys Henry Dick, No. 1,547,369, issued July 28, 1925, has been highly successful" in immunization against and prevention of scarlet fever, and has been widely used throughout the United States and foreign countries for that purpose. It has been heretofore administered by hypodermic injections which have sometimes been accompanied by difiiculties in administration and use.

We have found that due to the injection of various preparations for the prevention of other diseases, such as diphtheria and tetanus, that there is an increasing incidence of sensitization to proteins contained. in culture mediums, and that this sometimes interferes with subsequent immunizationagain'st scarlet fever by hypodermic injection of the toxin disclosed in the above mentioned patent. In certain individuals reactions such as vomiting-may occur following one or more of the hypodermic injections of the toxin due to the fact that it is not possible to increase the dosage gradually enough without making too many injections, and sometimes due to the fact that in the more dilute solutions for earlier injections in a given instance the toxin has deteriorated from not being used sufllciently soon after being diluted. I

In some instances, as where a patient is aflected with severe heart lesions or hemophilia, and who might be seriously affected by an attack of scarlet fever, there has been a reluctance on the-part of physicians to administer the toxin hypodermatically.

In immunization against scarlet fever hypodermatically it has been found advisable to extend the immunization over a period of approximately four weeks. In many instances it is highly desirable to lessen this immunicatlon period, especially in scarlet fever epidemics.

We so prepare and condition a toxin specific to scarlet fever that it may be readily administered by mouth, and with as satisfactory immunizing results as though injected hypodermatically, and without the difliculties incident to such injections and so that it maintains its potency for a longer period of time than-the toxin diluted for hypodermic injection.

After ascertaining that the toxin specific to scarlet fever may be absorbed in the blood stream from the alimentary tract and that it is not di-' the toxin was'so prepared and conditioned that the time required for its solution in the body of the patient is'l onger than the time during whichv toxin may be expected to normallyremain in the stomach and shorter than the time normally required forit to leave the small intestine, it may be administered orally with highly satisfactory immunizing results and without the difliculties incident to its being injectedhypodermatically and the dosage increased more gradually. when prepared in accordance with our invention the toxin may be administered orally at shorter intervals than when injected hypodermatically, and the period required for immunization may be substantially lessened.

Preparation of toxin 7 In order to obtain the proper toxin for immunization by mouth, hemolytic streptococci identified as scarlet fever streptococci through their production of the soluble toxin specific to scarlet fever are grown in a suitable culture medium. An illustration of a satisfactoryculture medium of low protein content is one which consists of a solution of- Percent Sodium chloride I 0.5 Wittes or protease peptone in distilled water. 1.0

venient concentration, is also prepared in distilled water, sterilized and allowed to cool. After cooling the broth is inoculated 'withpure cultures of hemolytic streptococci determined to be specific for scarlet fever, and the sugar solution.

is added in amounts up to 1%. This sugar solution may be added all at one time or in divided amounts during the incubation period.

The inoculated broth is placed in an incubator and maintained at 3 5-3'l centigrade.

The hemolytic streptococci produce acid during their growth in this medium. As we have found that scarlet fever toxin is destroyed by more transient exposure to chan-g'es in hydrogen ion concentration, we keep the reaction of the broth at about pH 7.0 throughout the incubation period in order to obtain a toxin of high potency and stability. This neutralization may be accomplished by hourly determinations of the reaction and addition of the indicated amounts s d y p p r ypsin, we found that when of a st rile solution of alkali such as sodium hy- 1 stance may be added in suitable amount.

droxide, or, more simply, by the addition of calcium carbonate powder to each container before 1 the broth is sterilized andfrequent shaking to keep the calcium carbonate distributed through- 3 out the broth during the period of growth.

After thirty-six to forty-eight hours, the growth Smears and plate cultures mine the purity of the cultures, any contaminated flasks being discarded and the rest saved 1 for concentration.

To stop further growth of the streptococci, with attendant production of acid, and to render the toxin containing broth free from livin scarlet fever streptococci, a germicidal or sterilizing subthis purpose phenol may be used in amount sufilcient to result in a 0.5% solution of phenol in the broth, or the streptococci maybe removed from the broth culture by filtration through a filter,

, such as a Berkefeld W, of such density that the streptococci do not pass through the filter. Our practice has usually been to add 0.5% phenol and then filter.

After the streptococci are killed, removed, or both, the toxin containing broth should be kept clean and free from bacteria, moulds or other 1 substances which might prove harmful when ingested, although it is not necessary to keep it sterile.

Concentration of the toxin This maybe done by the following or similar procedures:

1. The toxin containing broth may be concentrated or evaporated from the liquid state in a vacuum chamber in which the fluid is first frozen and then evaporated from the frozen state. If

the toxin is to be administered in the liquid state, it is not necessary to evaporate it to dryness, only enough evaporation being required to obtain a' sufiicient concentration of the toxin so that the required doses are contained in a small volume.

2. The toxin may be concentrated by adding to the toxin containing broth, a protein precipitating substance such as ammonium or sodium sulphate or alcohol in suitable amounts,'reserving the precipitate and drying it in, a dessicator'orredissolving the precipitate in distilled water and evaporating in a vacuum chamber from the frozen state. If the toxin is precipitated with a sulphate, care is taken that too much sulphate is not left in the precipitate. Thisma be accomplished by promptly blotting the wet precipitate between layers of filter paper, or by dialysis. If dialysis is employed, loss of toxin may be minimized by use of a membrane, such as Cellophane #1200.

cipitation whichwe have .found satisfactory is to j add to the chilled toxin containing broth three to four times its volume of chilled ethyl alcohol (95% or anhydrous), allowing the mixture to stand until the precipitate fiocculates, filtering and redissolving th precipitate in distilledwater, freezing the solution and evaporating it to a white, or almost white, substance in vacuum.

Evaporation may be conducted in the air but evaporation in a vacuum seems preferable because oxidization may destroy scarlet fever toxin.

is usually heavy and the cultures may be removed from the incubator.

may be made from each flask or .bottle to deter- For 0 diluted with inert and harmless substances and If evaporation is carried on at a low temperature there is less likelihood that some of the scarlet fever toxin may be destroyed.

When the concentrated toxin is obtained, it is standardized by means of testing various appropriate dilutions .(rendered free from bacteria by filtration through a suitable porcelain filter) by means of skin tests made by intradermal injection of 0.1 c. c. of each dilutionand comparing the resulting reactions with the reactions resulting in the same susceptible individuals from the intradermal injection of one standard skin test dose of scarlet fever toxin.

When the potency of the concentrated toxin .has been determined, it is ready for use in tab- Preparationof the concentrated toxin for oral administration If the concentrated toxin is employed in liquid form the doses are accurately measured and filled into suitable containers that are enteric sealed or coated.

If the dry form of concentrated toxin is employed, weighed portions of thepowder maybe filled into capsules or made into pills or tablets containing the required amounts of toxin by any convenient method suchas rolling or compress- One satisfactory method of preparing tablets from the dry toxin is as follows:

A weighed amount. of the standardized toxin, for example, 0.8 gram, is placed in a clean glass mortar and triturated' with pure lactose, 26

I rials have been employed. These coatings have grains, and best grade of acacia, 2 grains, until a homogeneous powder is formed. The material is then screened through a silk sieve andgranulated with 3 drops of distilled water. The granulated material is dried. in the open air at room temperature and 1 grain corn starch and 1 grain talc added. 2.12 grains of the resulting mixture is compressed into a tablet A1 inch in diameter.

If the dried toxin used contained 25,000,000 skin test dose of toxin per gram, each tablet made 'by this method should contain 1,000,000 skin test doses of scarlet fever toxin.

Th dry powder containing'the toxin with or I withput mixture with such' substances as starch,

lactose, talc or acacia may be filled into capsules or similar containers.

After the tablets, pills, capsules, perles or the like containing. the scarlet fever toxin, in either liquid or dry form, have been prepared they are ready for enteric coating or sealing.

Tablets, pills and capsules of scarlet fever toxin coated and sealed with a variety of enteric mateconsisted of substances such as the following used alone or in the various combinations.

Stearic acid, stearates, oleates, shellac suitable 'for food, ammoniated shellac, gelatine treated time required for solution of-the tablet whenplaced in fiuids such as water or watery solutions of weak acid is longer than the period of time the tablet may be expected to remain in the stomach under normal conditions, and shorter than the time normally required for the tablet to leave the small intestine.

The environmental coatings are largely composed of substances which dissolve more readily in the intestinal fluids than in the more acid fluids of the normal stomach; so that the tablets do not, as a rule, disintegrate until they reach the intestine. 1 a

. The term enteric is used to indicate a substance or substances which will largely prevent exposure of the medicament in the stomach and yet permit exposure of the medicament in'the'intestine.

One example of a satisfactory coating described as belongingto the group of timed coatings consists of a powdered mixture of fatty acid, wax

and hygroscopic vegetable components with a binding agent such as white shellac. A satisfactory form of it may be prepared by melting together 55.5 parts stearic acid, 24.25 parts carnauba wax and 1.75 parts of petrolatum. To this hot mixture are added 13.9 parts finely powdered agar and 4.6 parts finely powdered elm bark. The mixture is then allowed to cool and ground to a fine powder. In coating these tablets they may be placed in a revolving coating pan and a solution of white shellac suitable for food dissolved in 95% ethyl alcohol poured into the revolving pan in an amount sufficient to coat the tablets evenly. An adequate amount of the powdered material previously prepared is then added while the pan continues to rotate.- Due to the adhesiveness of the shellac, the tablets take up an even coating of the powder. The tablets are then the form of enteric coated tablets.

under normal conditions, the dosage of scarlet fever toxin required to immunize persons susceptible to scarlet fever is related to the speed of.

the tablets after they rach the In other immunization, in which the enteric coated tablets administered did not disintegrate in the buflere'd. salt solution until after eight to ten hours, the maximum daily dosage required to produce immunity to both one and two skin test doses of toxin injected intradermally was six to ten million skin test doses administered by mouth.

Attempts to change during the course of immunization from enteric coated tablets of a prolonged distintegration time to tablets requiring a shorter time for disintegration have resulted innausea and vomiting unless the dosage was reduced.

It has also been lea'rned'that the dosage of scarlet fever toxin administered by mouth in the form of enteric coated preparations for the purpose of immunizing persons susceptible to scarlet fever against that disease should be properly graduated in order to avoid unnecessary nausea,

placed on trays and flamed briefly on both sides.

A satisfactory example of an environmental enteric coating with which good results have been obtained in oral immunization against scarlet fever is a synthetic resin of the polyhydric alcohol polycarboxylic acid group. Such a resin may be prepared by fusing together stearic acid (17.57 parts), phthalic anhydrlde (53.65 parts) and glycerol (27.78) in an aluminum dish at 200 C. until the acid number has been reduced to 45. The resulting waxy resin is dissolved in a volatile solvent such as methyl propyl ketone and introduced in appropriate amounts into a rotating coating pan containing the tablets to be coated.

When enough of the solvent has been evaporated so that the tablets tend to stick together, any

aggregations are broken up and powdered talc is introduced into the rotating pan to assist in the drying process and prevent the tablets sticking together. When sufllciently dried, the tablets are removed from the pan and spread on trays while the drying is completed.

Regulation of dosage solutions containing the tablets may be agitated from time to time. If the tablets disintegrate in the acid solution at room or body temperature in less than three hours, they are apt to cause nausea in certain doses when administered to persons susceptible to scarlet fever. If the tablets are substantially insoluble in the stomach vomiting and diarrhoea. This similarly applies to the tablets without enteric coating.

. Because of the wide range in time required for solution or disintegration of the various enteric materials employed by the different manufacturers of biologic products, it is apparent that to.

obtain the greatest usefulness to the public of this simple method for preventing scarlet'fever, correct standardization of the toxin and the solubility of the enteric coated preparations to be furnished by the various manufacturers must be carefully controlled; further, the dosage should be related to the type of coating and proper graduation of thedosage should be assured.

We wish the term tablet" or tablets" as used in the specification and claims to be interpreted as including not only tablets but pills, perles, capsules or other'preparations suitable for administration by mouth,,containing scarlet fever toxin free from living scarlet fever streptococci, in either dry or liquid from.

The term concentrating is employed in the specification and in the claims to covernot only the evaporation of the toxin containing medium, whether under atmospheric 0r reduced'pressure, or from the liquidor the frozen state, to produce a concentrated toxin still in the liquid state, or a toxin containing solid, whether substantially 'dry or dried, but also the toxin in substantially dry or dried form when obtained by precipitation or by a combination of precipitation and evaporation The term "concentrated is used in the specification and claims to cover the product of any of the just defined concentrating methods.

Having now described our invention, we claim: 1. A tablet for oral administration comprising a toxin specific to scarlet fever derived from hemolytic streptococci and free from living scarlet fever streptococci, the toxin being of such potency that approximately one million skin test 4- doses is contained in the tablet substantially onefourth inch in diameter, and an enteric covering for protecting the toxin against absorption in the stomach but permitting its being absorbedwhile in the intestine when thus administered.

2. A tablet for immunizing against scarlet fever of a size suitable for oral administration containing a toxin specific to scarlet iever derived from-'hemolytic streptococci and free from fever streptococci, embodied ina tablet'of a size suitable for oral administration having a. potency' of at least one million skin test doses, and an enteric coating for] protecting the toxin against absorption by the..body until it reaches the intestines where it is released, when thus administered. p V a 4. An immunizing preparation for oral administration, comprising a concentrated powdered toxin specific to scarlet fever derived from hemolytic streptococci and free from'living scarlet fever streptococci, having a potency of approxi mately twenty-five million to seventy-five million skin test doses per g ram, in tablet form of a size suitable for-oral administration, such taigets being-of-the concentrated potency eas to produce immunity whenabsorbed in the intestinesv and covered'with an enteric coating which will prevent the toxin'being absorbed until-it reaches the intestines and permitting its beingflabsorbe while there, when administered orally.

5. A tablet for producing generaL- anti-toki immunity} against scarlet fever when administered orally, comprising a soluble bacterialtoxin Specific to scarlet fever derived from hemolytic streptococci and free .fromfliving scarlet fever streptococci, thevtoxin having a potency of at least twenty-five million skin test doses per gram of toxin with such tablets containing suii'icient toxin andbeing of such concentrated potency as to produce immunity when absorbed'in'the intestines, and an enteric covering for preventing-the toxin in the tablets from being absorbed as it passes from the mouth and through the stomach but permitting its being absorbed while in" the intestines, when thus administered.

' GEORGE F. DICK, GLADYS H. DICK.

JONATHAN E. WOOD. 

